The master sex-determination locus in threespine sticklebacks is on a nascent Y chromosome

BACKGROUND: Many different environmental and genetic sex-determination mechanisms are found in nature. Closely related species can use different master sex-determination switches, suggesting that these developmental pathways can evolve very rapidly. Previous cytological studies suggest that recently diverged species of stickleback fish have different sex chromosome complements. Here, we investigate the genetic and chromosomal mechanisms that underlie sex determination in the threespine stickleback (Gasterosteus aculeatus). RESULTS: Genome-wide linkage mapping identifies a single chromosome region at the distal end of linkage group (LG) 19, which controls male or female sexual development in threespine sticklebacks. Although sex chromosomes are not cytogenetically visible in this species, several lines of evidence suggest that LG 19 is an evolving sex chromosome system, similar to the XX female/XY male system in many other species: (1) males are consistently heterozygous for unique alleles in this region; (2) recombination between loci linked to the sex-determination region is reduced in male meiosis relative to female meiosis; (3) sequence analysis of X- and Y-specific bacterial artificial chromosome (BAC) clones from the sex-determination region reveals many sequence differences between the X- and Y-specific clones; and (4) the Y chromosome has accumulated transposable elements and local duplications. CONCLUSIONS: Taken together, our data suggest that threespine sticklebacks have a simple chromosomal mechanism for sex determination based on a nascent Y chromosome that is less than 10 million years old. Further analysis of the stickleback system will provide an exciting window into the evolution of sex-determination pathways and sex chromosomes in vertebrates.     

Changes in morphological characteristics of an introduced population of the threespine stickleback <Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis> in Lake Towada,northern Japan

A freshwater population of the threespine stickleback Gasterosteus aculeatus occurs at an elevation of 400m in Lake Towada in the mountains of northern Japan. Morphological characteristics of each sex of this population were studied between 1985 and 1992. The 1985 fishes were larger in standard length than even anadromous populations, as well as all other freshwater populations, so the large body size of this population was unusual for the species. The population was also characterized by large ratios of head length, eye diameter, and gill raker length, and a higher ovary volume in 1992 than in 1985, but it was not readily characterized by meristic characters. The population may have been introduced within the last 20 years (probably in the early 1980s). The large body size of the population may not reflect a defense against predators because coexisting fishes were not important predators on threespine stickleback adults. It is supposed that the change of body size was induced by environmental changes in prey abundance in the lake and/or by the longevity of fishes.     

Structure of the C-terminal RNA-binding domain of hnRNP D0 (AUF1), its interactions with RNA and DNA, and change in backbone dynamics upon complex formation with DNA

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP) -type RNA-binding domains (RBDs), each of which can specifically bind to the UUAG-sequence. hnRNP D0 also binds specifically to single-stranded d(TTAGGG)(n), the human telomeric DNA repeat. We have already reported the structure and interactions with RNA of the N-terminal RBD (RBD1). Here, the structure of the C-terminal RBD (RBD2) determined by NMR is presented. It folds into a compact alpha beta structure comprising an antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. In addition to the four beta-strands commonly found in RNP-type RBDs, an extra beta-strand, termed beta 4(-), was found just before the fourth beta-strand, yielding a five-stranded beta-sheet. Candidate residues of RBD2 involved in the interactions with RNA were identified by chemical shift perturbation analysis. Perturbation was detected on the beta-sheet side, not on the opposite alpha-helix side, as observed for RBD1. It is notable that the beta 4(-) to beta 4 region of RBD2 is involved in the interactions in contrast to the case of RBD1. The chemical shift perturbation analysis also showed that RBD2 interacts with DNA in essentially the same way as with RNA. Changes in the backbone dynamics upon complex formation with DNA were examined by means of model free analysis of relaxation data. In free RBD2, the beta 4(-) to beta 4 region exhibits slow conformational exchange on the milli- to microsecond time scale. The exchange is quenched upon complex formation. The flexibility of free RBD2 may be utilized in the recognition process by allowing different conformational states to be accessed and facilitating induced fit. Additionally, faster flexibility on the nano- to picosecond time scale was observed for loop 3 located between beta 2 and beta 3 in free RBD2, which is retained by the complex as well.     

Reduced early alcohol-induced liver injury in CD14-deficient mice

Activation of Kupffer cells by gut-derived endotoxin is associated with alcohol-induced liver injury. Recently, it was shown that CD14-deficient mice are more resistant to endotoxin-induced shock than wild-type controls. Therefore, this study was designed to investigate the role of CD14 receptors in early alcohol-induced liver injury using CD14 knockout and wild-type BALB/c mice in a model of enteral ethanol delivery. Animals were given a high-fat liquid diet continuously with ethanol or isocaloric maltose-dextrin as control for 4 wk. The liver to body weight ratio in wild-type mice (5.8 +/- 0.3%) was increased significantly by ethanol (7.3 +/- 0.2%) but was not altered by ethanol in CD14-deficient mice. Ethanol elevated serum alanine aminotransferase levels nearly 3-fold in wild-type mice, but not in CD14-deficient mice. Wild-type and knockout mice given the control high-fat diet had normal liver histology, whereas ethanol caused severe liver injury (steatosis, inflammation, and necrosis; pathology score = 3.8 +/- 0.4). In contrast, CD14-deficient mice given ethanol showed minimal hepatic changes (score = 1.6 +/- 0.3, p < 0.05). Additionally, NF-kappa B, TGF-beta, and TNF-alpha were increased significantly in wild-type mice fed ethanol but not in the CD14 knockout. Thus, chronic ethanol feeding caused more severe liver injury in wild-type than CD14 knockouts, supporting the hypothesis that endotoxin acting via CD14 plays a major role in the development of early alcohol-induced liver injury.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Synthesis and Properties of Dinucleoside Monophosphates Containing 2-Aminoadenine 8,2′-S- and Uracil 6,2′-O-Cyclonucleosides

Abstract

Two sequence isomers of dinucleoside monophosphates containing 8,2′-anhydro-2,6-diamino-8-mercapto-9-β-D-arabinofuranosylpurine (2NH₂A^s) and 6,-anhydro-6-hydroxy-1-ß-D-arabinofuranosyluracil (U^o), 2NH₂A^s pU^o (1) and U^o p2NH₂A^s (2) were synthesized by the phosphodiester method. Examination of the UV, CD and NMR spectra of these dimers led us to the conclusion that, whereas compound (1) did not take a stacked conformation, compound (2) took a well stacked conformation in which the bases were stacked along a left-handed screw axis. Both the dimers formed a complex with ethidium bromide.     

Purification and Properties of Acid Carboxypeptidase II from Aspergillus oryzae

Acid carboxypeptidase II from Aspergillus oryzae was purified from the rivanol non-precipitated fraction. The purified enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The optimum activity of the enzyme lay at pH 3.0 for carbobenzoxy-L-glutamyl-l-tyrosine. The enzyme was inhibited by diisopropylphosphorofluoridate and SH reagents such as p-chloromercuribenzoate and monoiodoacetate, but not by such metal chelating agents as ethylenediaminetetraacetate, α, α′-dipyridyl and o-phenanthroline. The molecular weight of the enzyme was estimated to be about 105,000.     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

A Novel Preparation of a trans-Fused Urea trans-3,4-Ureylenethiophane-1,1-dioxide

A novel synthesis of trans-3,4-ureylenethiophane-1,1-dioxide (X, R=H) from sulfolene via trans-3,4-diaminothiophane-1,1-dioxide (VII, R₁=R₂=H) is described.